Animals, cells and methods for production of detectably-labeled antibodies

ABSTRACT

Genetically-modified mammals and immune cells are provided which are capable of producing or secreting detectably-labeled immunoglobulin molecules as a result of genetic modifications of at least one immunoglobulin gene in the genome thereof, such that a fusion polynucleotide encoding a detectable protein or peptide and an immunoglobulin component molecule is present.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/982,120, filed Oct. 17, 2001, now U.S. Pat. No. 7,091,396, whichclaims priority under 35 U.S.C. § 119(e) to provisional application Ser.No. 60/241,053, filed Oct. 17, 2000, both of which—are incorporatedherein by reference in their entireties. Applicants claim the benefitsof these applications under 35 U.S.C. § 19(e) and 35 U.S.C. §120.

FIELD OF THE INVENTION

The invention is directed to methods for obtaining detectably-labeledantibodies to any antigen by using genetically-modified mammals whichexpress at least one detectably-labeled antibody molecule component.

BACKGROUND OF THE INVENTION

Antibodies are widely used for diagnostic and research purposes forlocalizing an antigen within a cell, tissue, or other biological sample.For histopathology or cytology, cells are usually first exposed to aprimary antibody which is specific for the desired target antigen butnot directly detectable. In a subsequent step, the primary antibody isdetected with a labeled secondary antibody which recognizes the primaryantibody. This procedure is somewhat cumbersome and may result inundesirable background staining due to non-specific reactivity of thesecondary antibody. In addition, in order to visualize two or moreantigens, the primary antibodies must be from two different species suchthat each secondary antibody is not cross-reactive; expensivespecies-specific secondary antibodies must therefore be used for eachprimary antibody. With advances in optical and digital imaging anddetection, multiply-labeled samples and simultaneous detection ofseveral analytes is possible, yet the aforementioned biologicallimitations do not permit full advantage to be taken of these advances.Moreover, since monoclonal antibodies are generated typically only inmice and rats, in conventional dual or triple labeling, only oneantibody can be monoclonal. This can be an important restriction.

Several techniques are available to circumvent these limitations,including direct chemical conjugation of the primary antibody with adetectable molecule, or direct conjugation with biotin and subsequencedetection with a fluorescent avidin. These procedures are cumbersome andrequires purification of the primary antibodies.

It is towards the facile preparation of a detectable primary antibody toany desired antigen that the present invention is directed.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

In one broad aspect, the invention is directed to a genetically-modifiedmammal capable of expressing at least one chimeric immunoglobulin genecomprising at least one detectable protein or peptide fused with a geneexpressing an immunoglobulin component selected from the groupconsisting of the kappa immunoglobulin light chain, the lambdaimmunoglobulin light chain, an immunoglobulin heavy chain, and anycombination thereof, wherein antibodies secreted by immune cells of thegenetically-modified mammal comprise said at least one detectableprotein or peptide. The immunoglobulin heavy chain gene may be IgG, IgM,IgD or IgA. In one embodiment, the at least one detectable peptide orprotein is present at the C-terminus of the gene product of the fusionpolynucleotide; preferably, the at least one detectable peptide orprotein present at the C-terminus of the gene product of the fusionpolynucleotide is located in exon G1. In another embodiment, the atleast one detectable peptide or protein is present at the C-terminus ofthe gene product of the fusion polynucleotide with a flexible linkerpeptide therebetween. Preferably, the least one detectable peptide orprotein present at the C-terminus of the gene product of said fusionpolynucleotide with a flexible linker therebetween is located in exonG1.

In one embodiment, the immunoglobulin molecule secreted by immune cellsof the above-mentioned genetically-modified mammal comprises at leastone detectable protein or peptide in the heavy chain of theimmunoglobulin molecule. In another embodiment, the immunoglobulinmolecule secreted by immune cells of the genetically-modified mammalcomprises at least one detectable protein or peptide in the light chainof said immunoglobulin molecule. In a further embodiment, theimmunoglobulin molecule secreted by immune cells of thegenetically-modified mammal comprises at least one detectable protein orpeptide in the heavy chain and at least one detectable protein orpeptide in the light chain of said immunoglobulin molecule.

In one embodiment of the invention, at least one of the aforementioneddetectable proteins or polypeptides is an autofluorescent protein orpeptide, a visibly-detectable protein or peptide, an enzymaticallyactive protein or peptide, a protein or peptide capable of interactingwith another molecule to produce a detectable product, or anycombination thereof. In another embodiment, the detectable protein orpolypeptide is capable of quenching or modulating fluorescence.Non-limiting examples of autofluorescent proteins or peptides includegreen fluorescent protein, red fluorescent protein, and a fluorescentanalog or fragment of any of the foregoing. Green fluorescent protein ispreferred. In another embodiment, the at least one detectable protein isa combination of an autofluorescent protein or peptide and anenzymatically-active protein or peptide, such as but not limited to acombination of green fluorescent protein and alkaline phosphatase.

In another broad aspect of the invention, a genetically-modified immunecell is provided which is capable of expressing at least one chimericimmunoglobulin gene comprising at least one detectable protein orpeptide fused with a gene expressing an immunoglobulin componentselected from the group consisting of the kappa immunoglobulin lightchain, the lambda immunoglobulin light chain, an immunoglobulin heavychain, and any combination thereof, wherein antibodies secreted by thegenetically-modified immune cell comprise the at least one detectableprotein or peptide. The immunoglobulin heavy chain gene may be IgG, IgM,IgD or IgA. In one non-limiting example, the at least one detectablepeptide or protein is present at the C-terminus of the gene product ofthe fusion polynucleotide; preferably, the polynucleotide encoding theat least one detectable peptide or protein present at the C-terminus ofthe gene product of said fusion polynucleotide is located in exon G1. Inanother embodiment, the at least one detectable peptide or proteinpresent at the C-terminus of the gene product of said fusionpolynucleotide has a flexible linker peptide therebetween; preferably,the polynucleotide encoding the at least one detectable peptide orprotein present at the C-terminus of the gene product of said fusionpolynucleotide with a flexible linker therebetween is located in exonG1.

The immunoglobulin molecule secreted by the aforementioned immune cellmay comprise at least one detectable protein or peptide in the heavychain of the immunoglobulin molecule, or it may comprise at least onedetectable protein or peptide in the light chain of said immunoglobulinmolecule. In another embodiment, the immunoglobulin molecule secreted bythe genetically-modified immune cells described above comprises at leastone detectable protein or peptide in the heavy chain and at least onedetectable protein or peptide in the light chain of the immunoglobulinmolecule.

The aforementioned genetically-modified immune cell may have at leastone detectable protein or polypeptide that is capable of quenching ormodulating fluorescence; or the protein or peptide is an autofluorescentprotein or peptide, a visibly-detectable protein or peptide, anenzymatically active protein or peptide, a protein or peptide capable ofinteracting with another molecule to produce a detectable product, orany combination thereof. In the example wherein the at least onedetectable protein is an autofluorescent protein or peptide, it may be,by way of non-limiting example, green fluorescent protein, redfluorescent protein, or a fluorescent analog or fragment of any of theforegoing. Green fluorescent protein is preferred. In anotherembodiment, the at least one detectable protein is a combination of anautofluorescent protein or peptide and an enzymatically-active proteinor peptide, such as but not limited to a combination of greenfluorescent protein and alkaline phosphatase.

The invention is also directed to a hybridoma comprising thegenetically-modified immune cell as mentioned above.

In yet another aspect, the invention is directed to a chimeric,detectably-labeled immunoglobulin molecule comprising at least onedetectable protein or peptide fused with the kappa immunoglobulin lightchain, the lambda immunoglobulin light chain, an immunoglobulin heavychain, or any combination thereof. The at least one detectable peptideor protein may be present at the C-terminus of the gene product of saidfusion polynucleotide, preferably located in exon G1. A flexible linkerpeptide may be provided therebetween. In another embodiment, apolynucleotide encoding said at least one detectable peptide or proteinpresent at the C-terminus of the gene product of said fusionpolynucleotide with a flexible linker therebetween is located in exonG1. The chimeric, detectably-labeled immunoglobulin molecule may have animmunoglobulin heavy chain gene is selected from IgG, IgM, IgD and IgA.At least one detectable protein or peptide may be present in the heavychain of said immunoglobulin molecule, the at least one detectableprotein or peptide may be present in the light chain of saidimmunoglobulin molecule, or, in a further embodiment, the at least onedetectable protein or peptide may be present in the heavy chain and atleast one detectable protein or peptide in the light chain of saidimmunoglobulin molecule.

The aforementioned chimeric, detectably-labeled immunoglobulin moleculemay have at least one detectable protein or polypeptide that is capableof quenching or modulating fluorescence; or, the at least one detectableprotein or peptide is an autofluorescent protein or peptide, avisibly-detectable protein or peptide, an enzymatically active proteinor peptide, a protein or peptide capable of interacting with anothermolecule to produce a detectable product, or any combination thereof. Inone embodiment, the at least one detectable protein is anautofluorescent protein or peptide, such as but not limited to greenfluorescent protein, red fluorescent protein, or a fluorescent analog orfragment of any of the foregoing. Preferably, it is green fluorescentprotein. In another embodiment, the at least one detectable protein maybe a combination of an autofluorescent protein or peptide and anenzymatically-active protein or peptide, such as but not limited to acombination of green fluorescent protein and alkaline phosphatase.

In a further broad aspect, the present invention provides a method forproducing a quantity of detectably-labelled polyclonal antibodies bycarrying out at least the steps of

-   -   a) providing a genetically-modified mammal as described        hereinabove;    -   b) immunizing the genetically-modified mammal with a preselected        immunogen, wherein the genetically-modified mammal generates        antibodies to the immunogen, wherein antibodies secreted by        immune cells of the genetically-modified mammal comprise the at        least one detectable protein or peptide; and    -   c) isolating the detectably-labelled antibodies from the        genetically-modified mammal.

The invention is also directed to a method for producing a quantity ofdetectably-labelled monoclonal antibodies comprising the steps of

-   -   a) preparing a genetically-modified mammal in accordance with        the above description;    -   b) immunizing the genetically-modified mammal with a preselected        immunogen, wherein immune cells of the genetically-modified        mammal generate antibodies to the immunogen, wherein antibodies        secreted by the immune cells comprise the at least one        detectable protein or peptide;    -   c) immortalizing antibody-producing immune cells isolated from        the genetically-modified mammal;    -   d) selecting immortalized immune cells isolated from the        genetically-modified mammal that secrete antibodies specific to        the immunogen; and    -   e) preparing a quantity of detectably-labeled monoclonal        antibodies from the selected immune cells.

In a further aspect, the invention is also directed to agenetically-modified mammal capable of producing a detectably-labeledimmunoglobulin in response to immunization by an antigen, the genome ofthe mammal comprising at least one fusion polynucleotide consisting of apolynucleotide sequence encoding at least one detectable protein orpeptide fused with a the kappa immunoglobulin light chain gene, thelambda immunoglobulin light chain gene, an immunoglobulin heavy chaingene, or any combination thereof, wherein antibodies secreted by immunecells of the genetically-modified mammal comprise the at least onedetectable protein or peptide.

In a further aspect, the invention is directed to a chimeric,detectably-labeled immunoglobulin molecule comprising at least onefluorescent protein or peptide and at least one fluorescence-quenchingor -modulating protein or peptide fused with a component of theimmunoglobulin molecule independently selected from either the kappaimmunoglobulin light chain, the lambda immunoglobulin light chain, animmunoglobulin heavy chain, or any combination thereof.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the beta-galactosidase-binding activity of a chimericfusion immunoglobulin secreted from 293T cells transfected with nucleicacid encoding a green-fluorescent-protein-labeled heavy immunoglobulin(IgG2) chain and a green-fluorescent-protein-labeled light (kappa) chainhybrid anti-beta-galactosidase immunoglobulin.

FIGS. 2 A-B show the fluorescence of antibodies in serum from kappa-GFPknock-in mice bound to Protein A/G beads, as compared to those fromwild-type mice.

FIG. 3 A-B demonstrate using PCR amplification that mice of theinvention may be homozygous or heterozygous for the kappa-GFP gene,using kappa light chain primers (FIG. 3A). Use of GFP primers (FIG. 3B)confirm the presence or absence of the GFP insert.

DETAILED DESCRIPTION OF THE INVENTION

The inventors herein have discovered a novel and facile method forpreparing useful quantities of detectably-labeled polyclonal ormonoclonal antibodies to any preselected immunogen. The method sparesthe need to perform any modification of the immunogen-specific (primary)antibody to provide a detectable label thereon, such as conjugation to afluorophore or enzyme, as the detectably-labeled antibodies of theinvention are inherently labeled as they are expressed and secreted fromgenetically-modified immune system cells. In its broadest aspect, theinvention provides genetically-modified animals and immune cells fromsuch animals in which the constant region of the heavy and/or the lightchain components of the antibodies produced by the animals or cells isfused with a detectable polypeptide, such as a fluorescent polypeptide,these fusion protein(s) resulting from the expression of a geneticmodification of the heavy and/or light chain genes wherein thepolynucleotide encoding the detectable label is incorporated into therespective genes. Such genetically-modified animals or cells areprovided such that any antibody expressed and secreted by the animal orcell comprises at least one labeled constant region. Thus, the mereinduction of a humoral immune response generates the desireddetectably-labeled antibody.

The detectable labels of the invention are polypeptides, such asproteins or peptides, and they may be introduced into the genome of amammalian organism by such methods as homologous recombination andtransfection, but such methods are not intended to be limitingwhatsoever, and one of skill in the art may prepare the construct(s) andmodified cells in any appropriate manner. Embryonic stem cells may be somodified such that the genome of the animal resulting therefromcomprises the genetic modification. Several examples provided below arefor illustrative purposes only.

The detectable polypeptide may be, by way of non-limiting example, afluorescent polypeptide, a visibly-colored polypeptide, a polypeptidewith enzymatic activity, or a polypeptide capable of interacting with orbinding to another molecule to produce a detectable product. Preferredare labels which require no direct interaction or further sampleprocessing for detectability, and thus are detectable by the applicationof exogenous methods such as absorbance of light, fluorescence, etc.More preferred are fluorescent polypeptides; most preferred is greenfluorescent protein and its polypeptide relatives.

Moreover, a plurality of detectable labels may be present in theantibodies produced by the animals or cells of the invention. Forexample, both a fluorescent label and an enzyme label may be appended intandem to the C-terminus of an immunoglobulin heavy chain gene, suchthat the resulting expressed and assembled immunoglobulin molecule isdetectable both by fluorescence and by histochemistry, by use of afluorigenic and chromogenic/precipitating substrate of the enzyme,respectively. In another example, the antibody is detectablefluorometrically and by Western blot. As noted above, either or both theheavy and light chain constant regions may be independently modified asdescribed herein, both with the same single or plurality of labels, or,for example, the heavy chain with a fluorescent label and the lightchain with an enzyme label. These particular examples of multiple labelson multiple sites of the antibodies are merely illustrative of the rangeof directly-labeled antibodies that the skilled artisan may be directedto prepare following the teachings herein, and any particular example orembodiment is not intended to be limiting whatsoever.

Examples of fluorescent polypeptides include but are not limited togreen fluorescent protein and other related polypeptide fluorophoreswhich may produce other colors; the availably of individually-detectableprimary antibodies in a mixture allows for the simultaneous localizationor quantitation of multiple target antigens or analytes in a biologicalsample. The green fluorescent protein of Aequorea victoria isparticularly preferred as the fluorescent protein. A cDNA for theprotein has been cloned (D. C. Prasher et al., “Primary structure of theAequorea victoria green-fluorescent protein,” Gene (1992) 111:229-33.).Aequorea green fluorescent protein (“GFP”) is a stable,proteolysis-resistant single chain of 238 residues and has twoabsorption maxima at around 395 and 475 nm; Excitation at the primaryabsorption peak of 395 nm yields an emission maximum at 508 nm with aquantum yield of 0.72-0.85 (O. Shimomura and F. H. Johnson J. Cell.Comp. Physiol. 59:223 (1962); J. G. Morin and J. W. Hastings, J. Cell.Physiol. 77:313 (1971); H. Morise et al. Biochemistry 13:2656 (1974); W.W. Ward Photochem. Photobiol. Reviews (Smith, K. C. ed.) 4:1 (1979); A.B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995); D. C. PrasherTrends Genet. 11:320-323 (1995); M. Chalfie Photochem. Photobiol.62:651-656 (1995); W. W. Ward. Bioluminescence and Chemiluminescence (M.A. DeLuca and W. D. McElroy, eds) Academic Press pp. 235-242 (1981); W.W. Ward & S. H. Bokman Biochemistry 21:4535-4540 (1982); W. W. Ward etal. Photochem. Photobiol. 35:803-808 (1982)). Mutants of GFP areembraced herein as they provide certain other characteristics, such asmutation of Serine 65 to Thr (S65T) simplifies the excitation spectrumto a single peak at 488 nm of enhanced amplitude (R. Heim et al. Nature373:664-665 (1995)), which no longer gives signs of conformationalisomers (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995)). Inanother example, U.S. Pat. No. 6,077,707 describes a nucleic acidmolecule comprising a nucleotide sequence encoding a functionalengineered fluorescent protein whose amino acid sequence issubstantially identical to the amino acid sequence of Aequorea greenfluorescent protein but differs by at least a substitution at T203 and,in particular, T203X, wherein X is an aromatic amino acid selected fromH, Y, W or F. In one embodiment therein, the amino acid sequence furthercomprises a substitution at S65, wherein the substitution is selectedfrom S65G, S65T, S65A, S65L, S65C, S65V and S651. In another embodiment,the amino acid sequence differs by no more than the substitutionsS65T/T203H; S65T/T203Y; S72A/F64L/S65G/T203Y; S65G/V68L/Q69K/S72A/T203Y;S72A/S65G/V68L/T203Y; S65G/S72A/T203Y; or S65G/S72A/T203W. In anotherembodiment, the nucleotide sequence encoding the protein differs fromthe nucleotide sequence of native green fluorescent protein by thesubstitution of at least one codon encoding an amino acid substitutionat L42, V61, T62, V68, Q69, Q94, N121, Y145, H148, V150, F165, I167,Q183, N185, L220, E222 (not E222G), or V224. In another embodiment, theamino acid substitution is: L42X, wherein X is selected from C, F, H, Wand Y, V61 X, wherein X is selected from F, Y, H and C, T62X, wherein Xis selected from A, V, F, S, D, N, Q, Y, H and C, V68X, wherein X isselected from F, Y and H, Q69X, wherein X is selected from K, R, E andG, Q94X, wherein X is selected from D, E, H, K and N, N121X, wherein Xis selected from F, H, W and Y, Y145X, wherein X is selected from W, C,F, L, E, H, K and Q, H148X, wherein X is selected from F, Y, N, K, Q andR, V150X, wherein X is selected from F, Y and H, F165X, wherein X isselected from H, Q, W and Y, I167X, wherein X is selected from F, Y andH, Q183X, wherein X is selected from H, Y, E and K, N185X, wherein X isselected from D, E, H, K and Q, L220X, wherein X is selected from H, N,Q and T, E222X, wherein X is selected from N and Q, or V224X, wherein Xis selected from H, N, Q, T, F, W and Y. These examples are merelyillustrative of the wide selection of fluorescent polypeptides and theircorresponding polynucleotide sequences that may be employed in thepreparation of the genetically-modified cells or animals of theinvention.

Another example of a fluorescent protein is red fluorescent protein fromcoral (Matz et. al., 1999, Nature Biotechnology 17:969). Fluorescentpeptides or fluorescent protein fragments of these and other fluorescentproteins are also embraced herein. In another example of a detectablepolypeptide, polypeptide labels capable of being detected include thosecontaining four cysteines at the i, i+1, i+4, and i+5 positions (i.e.,WEAAAREACCRECCARA (SEQ ID NO: 1)). These peptides will bind specificallyto the fluorescein derivative4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FLASH), which isnon-fluorescent until bound. The use of such peptides has the advantageof providing fusion proteins of much smaller size than GFP, andfluorescence is easily detected by addition of FLASH (Griffin et. al.,1998, Science 281:269). Thus, the use of polypeptides that are capableof specifically binding a detectable label are embraced herein asanother embodiment of the present invention.

Examples of polypeptides capable of being detected by enzymatic activityinclude various enzymes and catalytic polypeptide fragments thereof.Particularly preferred enzymatic labels include those which are able toproduce a detectable color or fluorophore in a single step requiring aminimum or reagents, such as alkaline phosphatase, which can cleave achromogenic substrate, such as p-nitrophenyl phosphate, or a fluorigenicsubstrate, such as ECF substrate (Amersham/Pharmacia); and a fluorigenichorseradish peroxidase substrate, FluoroBlot (Pierce Chemical Co.).Another example is a fluorescent beta-galactosidase substrate that canbe used in live cells and is 100-fold more sensitive than GFP (Zlokarniket. al., 1988, Science 279:85). The skilled artisan by the teachingsherein will be amply aware of polynucleotides that may be fused to theimmunoglobulin constant region(s) and upon expression produce anenzymatically-active fusion polypeptide comprising the immunoglobulinheavy and/or light chain.

Such labels may also result in the precipitation of a substrate, forhistochemical localization of the antibody, or the label may interactwith another detectable component, ands thus be, for example, an intein,a biotin-binding subunit of streptavidin or avidin, a His tag, or achitin-binding domain.

Guidance for the selection of the immunoglobulin genes and locationstherein in which to fuse the detectable polypeptide(s) of the inventionmay be performed as follows. As is well known, the antibody molecule iscomposed of two heavy chains and two light chains. Each chain has a twoidentical variable regions which bind antigen, and a constant region.The N-terminus of both chains is part of the variable region and theC-terminus is part of the constant region.

The genomic organization of heavy chain contains a number of repeats ofV, D, and J regions upstream of several constant regions. The differentconstant regions define the eventual antibody class (e.g., IgG1, IgG2a,IgA, etc). During B-cell maturation, a single V, D, and J are joined toform a single exon. Additionally, late in B-cell development, a singleconstant region is chosen in a process called “class switching”. Eachconstant region contains a number of exons. After transcription, the VDJand constant regions are spliced into the mature mRNA.

To generate the fusion protein of the invention, a polynucleotideencoding the detectable polypeptide is inserted into the constantregion, or preferably, is appended to portion of the gene resulting inthe expression of the fused polypeptide label on the C-terminus of theconstant region of the light and/or heavy chains, optionally with aflexible linker peptide.

There are two classes of light chains, kappa and lambda. Kappa comprises95% of antibodies, lambda 5%. Furthermore, there are two forms of eachclass of the heavy chain—a secreted and a membrane bound form. Themembrane bound form is responsible for signaling to the B-cell when itis crosslinked by an antigen and is required for B-cell maturation andantibody production. The two forms result from alternative splicing.Preferably, only the secreted form is modified as described herein. Themembrane bound form would be completely wild type, to maintain fidelityof generation of the antibody response.

Thus, an immune cell with the genetic modifications described herein maysecrete immunoglobulin that may have possess a detectable polypeptidelabel fused in the heavy chain constant region, or a detectablepolypeptide label fused in the light chain constant region, or theimmunoglobulin may have both a heavy and light chain fusion component.Moreover, each detectable label may be a single detectable label or aplurality of labels, in tandem or not, optionally separated from theimmunoglobulin portion of the polypeptide by one or more linkersequences. The label on the heavy chain may be the same or differentfrom that on the light chain, for immunoglobulins that have both heavyand light chains as fusion products. Although the placement of thedetectable label is preferably on or near the C-terminus, the fusedlabel(s) may be at any position(s) with does not detract from theability of the immunoglobulin to interact with and bind to its targetantigen. The invention herein embraces any and all variations in theposition, placement, linkers, and number of fused peptides orpolypeptides, with the object of the invention to provide at least onedetectable label on an immunoglobulin molecule generated by exposure ofan animal or immune cell with the aforementioned genetic modification toany immunogen capable of eliciting secretion of an antibody directedthereto.

As described above, a mammalian organism, such as a mouse, rat, rabbit,goat, cow, horse, may be provided with the genetic modification asdescribed herein. Example 4 below demonstrates the invention using mice,in which endogenous production of fluorescent antibodies is shown.Exposure of the animal to a preselected immunogen will elicit anantibody response, the secreted antibodies having the detectablelabel(s) present in every antibody molecule. For production ofpolyclonal antibodies, antiserum from the immunized animals may becollected, the antibodies purified if desired, for subsequent use insuch areas as histochemistry, diagnostics, etc.

If detectably-labeled primary monoclonal antibodies are desired, routinemethods may be followed using an animal, such as a mouse, with thegenetic makeup hereindescribed. B-cells secreting the detectably-labeledprimary antibodies may be fused for immortalization, and screening andselection for stable antibody-secreting hybridomas obtained by routinemethods.

Of course, detectably-labeled primary anti-idiotype antibodies may beprepared by the foregoing methods, using antibodies as immunogen.

As mentioned above, the polyclonal and monoclonal antibodies generatedby the methods described herein have several advantages, including 1)because the primary antibody is endogenously fluorescent, no secondaryantibody or chemical labeling is required, 2) autofluorescent proteinsare currently available in several different colors, including blue,cyan, green, yellow, and red, allowing for simultaneous labeling ofseveral antigens, and simultaneous detection by instrumentation capableof discriminating several fluorophores simultaneously. In addition, theavailability of two different means for detection, such as but notlimited to a visibly-detectable and enzymatically-detectable marker,provides detectability under a variety of conditions, applicable tovarious research and diagnostic applications, among others.

Introduction of the genetic modification(s) described herein into anembryonic stem cell or other cell type may be achieved by a variety ofprocedures known in the art. Preferably, the genetic modification isintroduced by a “knock-in” procedure in which the wild-typeimmunoglobulin gene(s) are replaced by the detectable-label-modifiedgenes. Such procedures may include the use of a bacterial artificialchromosome, as exemplified in Example 2 below, although this example ismerely illustrative and non limiting as to procedures for achieving thegenetically-modified mammal or mammalian cells of the invention.

Antigens to which detectable antibodies may be raised ingenetically-modified animals or immune cells as described herein are notlimited to any particular types or classes of immunogen, and includesthose for which a detectable antibody is desirable. Such antigenscomprise a vast list. By way of non-limiting example, this includesdiagnostic and research reagents for identifying the presence of and/orquantitating various medically-important biomolecules in bodily fluids,biopsy and necropsy samples, for example, all diagnostic tests whichemploy immunoassay protocols, including ELISA, radioimmunoassay, EMIT,immunofluorescence, fluorescence polarization, and other methods fordetecting the interaction between and biomolecule of interest and anantibody thereto. Medical diagnostics include, by way of non-limitingexample, assays for autoimmune disorders, cardiovascular disorders,diabetes, endocrine disorders, fungal, bacterial, viral, parasitic, andother infectious agents, hematologic diseases, immunologic diseases,hepatic diseases, oncologic diseases, thyroid diseases, and toxicologyand drugs of use and abuse. Moreover, as mentioned above, the ability todiscriminate between differently labeled primary antibodies without theneed for, and concomitant disintegration of signal, by multiplesecondary antibodies, will permit multiple analytes to be measuredsimultaneously, decreasing the costs and increasing the amount ofinformation available for rendering diagnostic and therapeuticdecisions. In the research area, innumerable new and known biomoleculesto which antibodies are routinely raised for identifying the location,movement, transport, role, etc., of such new or rediscoveredbiomolecules in biological processes will be simplifiable, as well aspermit the simultaneous and facile monitoring of multiple biomoleculesusing differently-labeled antibodies to several biomoleculesparticipating in a process. The discussion herein on particular antigensis not intended to be at all limiting but is merely illustrative of someexamples of the utility of the invention.

In another example of the invention, a genetically-modified mammal maybe prepared which responds to immunization with an antigen by theproduction of chimeric immunoglobulin molecules capable of reading out asignal or altering the signal produced only on binding with the targetantigen. This signal may be, for example, an increase, decrease orchange in fluorescence. Such chimeric antibodies may be used in a verysimple homogeneous immunoassay in which, on combining with a sample,indicates the presence or extent of the level of the antigen in thesample. Pairs of proteins or peptides capable of undergoing suchmodulation in fluorescence include FRET pairs, such as described, forexample, in U.S. Pat. No. 5,998,204, incorporated herein by reference inits entirety.

Such chimeric antibodies are prepared by following the methods describedherein. In the example wherein the fluorescence is detected, the genesencoding the chains of the secreted immunoglobulin are modified at thepolynucleotide level to provide that both a fluorescent peptide orpolypeptide, and a fluorescence-quenching or -modulating peptide orpolypeptide, are fused into either the heavy immunoglobulin chain or thelight immunoglobulin chain in the appropriate position in the genome ofthe mouse. The positions of integration into the respectiveimmunoglobulin components are such that the proximity of the fluorescentpolypeptide and the fluorescence-quenching or -modulating polypeptide inthe secreted, whole immunoglobulin molecule, change on binding of theimmunoglobulin to its target antigen. This change in proximity altersthe interaction between the fluorophore and the quencher or modulatorresulting in a modulation in the detectable fluorescence on exposure ofthe immunoglobulin to its excitation wavelength. Such positions areselected to not alter the ability of the complete immunoglobulinmolecule to assemble, be secreted, or bind the antigen. These selectionsare within the realm of the skilled artisan. Known fluorescent peptidesor polypeptides as well as peptides and polypeptides capable ofquenching or modulating the fluorescence, such as FRET pairs, are knownand can be selected to provide the chimeric immunoglobulin, and mammalscapable of producing the immunoglobulin after immunization.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1 Secretion of Detectably-Labeled Antibodies

A rat IgG2A antibody hybridoma which produces a monoclonal antibodyreactive against beta-galactosidase was used to determining whether GFPfusion proteins would be correctly assembled, secreted, and recognizeantigen. The cDNA of the secreted form of the heavy and light chainsagainst beta-galactosidase were cloned. The polynucleotide sequenceencoding GFP was placed in front of the stop codon of both the heavychain of IgG2A and light kappa chains. The plasmid combinations of 1)non-GFP tagged, 2) IgG2A heavy chain tagged, 3) light chain tagged, and4) both heavy chain and light chain tagged, were transfected into 293Tkidney cells and tested for ability to bind antigen.

The 293T cells do not express any endogenous antibodies. They weretransfected with two expression vectors of combinations of IgG2A andkappa chains against beta-galactosidase: 1) IgG2A and kappa; 2) IgG2Aand kappa-GFP; 3) IgG2A-GFP and kappa; and 4) IgG2A-GFP and kappa-GFP.The transfected cells will express and assemble and secrete the completeantibody. Forty-eight hours after transfection, the medium washarvested. This contained unpurified antibodies which was used for theexperiments below

Confirmation of the secretion of the detectably-labeled antibody wasobtained in several experiments. In one experiment, cells were fedradiolabeled ³⁵S methionine after transfection and newly synthesizedproteins were labeled. The secreted proteins were collected. Twofractions were evaluated, one that bound to Protein G and one that boundto beta-galactosidase immobilized on beads. Protein G is a toxin whichbinds antibodies. As shown in FIG. 1, both GFP-tagged and untaggedantibodies bound to Protein G and to beta-galactosidase. The affinityand specificity towards beta-galactosidase appears greater. Thus, theantibody recognizes antigen and is correctly folded. When only lightchain or heavy chain but not both are transfected, the chains did notbind to either protein G or beta-galactosidase. When examined under themicroscope, the beads where bright green, showing that the GFP iscorrectly folded and fluorescent.

EXAMPLE 2 Preparation of Genetically-Modified (Knock-In) AnimalsComprising Detectably-Labeled Immunoglobulin Genes

Knock-in or gene replacement involves replacing an endogenous piece ofDNA in the chromosome with a constructed piece of DNA. Thus, theconstructed DNA must correctly integrate into the same genetic locationas the gene to be replaced. This is distinctly different from transgenictechnology where a constructed DNA is randomly introduced into a cell.

I. Construction of a segment of bacterial plasmid DNA that contains thechange of interest and extensive homology to the target. Generation ofthe IgG1-GFP fusion knock-in construct is described below. The VDJvariable regions of the IgG1 constant chain are far upstream (5′) ofthis area. Immediately upstream of this area are the IgM and IgDconstant regions. Downstream to this area are the constant regions forIgG2a, IgG2b, IgG3, IgE, and IgA. The exons CH1, CH2 and G1 are splicedtogether to make the constant region of the secreted form. To make themembrane bound form, CH1, CH2, G1 (except the last 2 amino acids), TD1,and TD2 are spliced together. In the targeting vector, GFP is fusedin-frame at the end of the G1 exon with a five-amino-acid linker ofGly-Gly-Ser-Gly-Gly (SEQ ID NO: 2) in-between. The membrane bound formsplices out the last two amino acids of G1 and thus the entire GFP.Therefore, GFP will be fused only to the secreted form.

A bacterial artificial chromosome (BAC) containing the entire IgG regionis obtained. The targeting vector contains the 5′ homologous regiondirectly PCRed from the BAC. The G1-GFP fusion is generated by primeroverlap extension PCR (Horton et al., 1990; Horton, 1995). A neomycinresistance gene is introduced as a selection marker. This neomycin geneis flanked on each side by a loxP site. Thus, after selection, the geneis deleted by Cre-mediated recombination. This eliminates problems duethe presence of the neomycin gene and its associated promoter (Zou etal., 1993). A 3′ homologous region is also PCRed from the BAC. Finally,a toxic gene, diphtheria toxin (DTA) is introduced just outside the areaof homology. Homologous recombination between the targeting vector andthe targeting region cuts out the toxin gene. Non-homologous randomintegration events often retain DTA and the cells will be killed (Yagiet al., 1990; Yagi et al., 1993; McCarrick, III et al., 1993).

After preparation, embryonic stem cells are transfected with thetargeting vector and selection with neomycin analog G418 is performed.Clones are screened for correct integration by Southern analysis andPCR. Subsequently, correctly targeted ES cell are injected intoblastocysts and implanted into mothers. The blastocysts develop intochimeric mice, where some cells are developed from the injected EScells. The chimeras are used to parent heterozygote mice. Twoheterozygous mice are then parented to produce homozygous mice.

The mouse IgG1 heavy chain and kappa light chains have been previouslytargeted for gene replacement. In those cases, the constant chain wasreplaced with human constant chains and the resulting mice generatedhumanized antibodies (Zou et al., 1993; Zou et al., 1994).

Production of Monoclonal Antibodies. Mice as prepared above areinoculated with the antigen of interest. After several weeks, theantibody production against the antigen is screened, as traditionallydone with an ELISA, where the antigen is immobilized, the mouse serum isadded, and an enzyme-linked secondary antibody against the primary isadded. The presence of enzymatic activity indicates that the mouse hasproduced specific antibodies. Since here, the antibodies are alreadyfluorescent, an ELISA is unnecessary and measurement of boundfluorescence is sufficient. Fusion of splenocytes from a positive mousewith myeloma cells is used to produce immortalized antibody-producingcells, following standard protocols. Further screening of colonies canbe performed using simple fluorescence measurements.

Antibody may be produced in quantity by growth of cell-hybrids,following standard protocols.

EXAMPLE 3 Homogeneous Immunoassays Using Detectably-Labeled Antibodiesfrom a Genetically-Modified Mouse

In a further example of the methods described in Example 2, above, agenetically-modified mouse may be prepared which responds toimmunization with an antigen by the production of chimericimmunoglobulin molecules capable of reading out a signal only on bindingwith the target antigen. Such chimeric antibodies may be used in a verysimple homogeneous immunoassay in which, on combining with a sample,indicates the presence or extent of the level of the antigen in thesample.

Such chimeric antibodies are prepared by following the methods herein.Both a fluorescent peptide or polypeptide, and a fluorescence-quenchingpeptide or polypeptide, are fused into either the heavy immunoglobulinchain or the light immunoglobulin chain in the appropriate position inthe genome of the mouse. Such pairs may include FRET pairs or proteins,such as described, for example, in U.S. Pat. No. 5,998,204, incorporatedherein by reference in its entirety. The positions of integration intothe respective immunoglobulin components are such that the proximity ofthe fluorescent polypeptide and the fluorescence-quenching polypeptidechange on binding of the immunoglobulin to its target antigen. Suchpositions also do not alter the ability of the complete immunoglobulinmolecule to assemble, be secreted, or bind the antigen. Both thefluorescent and the fluorescence-quenching protein or peptides may befused into the same immunoglobulin component, at positions whereinantigen binding induces a conformational change and thus a proximitychange among the pair and attendant modulation of fluorescence.

Examples of fluorescent peptides and proteins are described above.Suitable pairs, for example include a blue-shifted GFP mutant P4-3(Y66H/Y145F) as the donor, and an improved green mutant S65T canrespectively serve as a donor and an acceptor for fluorescence resonanceenergy transfer (FRET; Tsien et al., 1993, Trends Cell Biol. 3:242-245).A genetically-modified mouse expressing antibodies with thesemodifications was immunized with human chorionic gonadotropin (hCG). Thefluorescence of antibodies produced by this mouse was increased uponbinding to hCG. This reagent was used in a simplified assay in anautomated fluorescence-based instrument for determining pregnancy amonga large battery of other diagnostic tests on blood and urine samples.

EXAMPLE 4 Kappa-GFP Knock-In Mice Produce Fluorescent EndogenousAntibodies

Kappa-GFP knock-in mice were prepared in accordance with the methodsdescribed in Example 2, above. At 8 weeks of age, animals were bled andserum was incubated with Protein A/G beads, to which antibodies in theserum bind. As shown in FIG. 2, under fluorescence illumination thebeads showed the characteristic fluorescence of GFP (FIG. 2A), whereasserum from wild-type mice incubated with Protein A/G beads did not (FIG.2B). Thus, the mice produced fluorescently-labeled, endogenousimmunoglobulin molecules, comprising kappa-GFP, fluorescent lightchains.

EXAMPLE 5 Confirmation of Genotype Using PCR for Kappa and GFP

FIG. 3 shows the results of PCR amplification using primers from up- anddown-stream of the section of the kappa light chain where the GFP wastargeted (FIG. 3A), or using GFP primers (FIG. 3B), from DNA extractsfrom a number of mice prepared in accordance with the present invention.The segment amplified from wild type kappa light chains is the smallestand runs the fastest on the gel (as indicated). The segment amplifiedwith the GFP insert is larger and migrates slower. In FIG. 3A, using thekappa primers, lane 2 has only the faster moving product and istherefore from a mouse that is homozygous for wild-type kappa lightchain. Lanes 3, 5, and 7-12 have both products, which indicates that themice are heterozygous. Lanes 1, 4, and 6 only have the slower movingproduct indicating that they are homozygous for the kappa with theinserted GFP. FIG. 3B shows using primers from the GFP coding regionthat lane 2 was negative for GFP, consistent with the observation thatthis mouse did not produce a PCR product from kappa-GFP. Lanes 1 and2-12 were positive for GFP, consistent with the results from the toppanel.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

-   Horton, R. M. (1995). PCR_mediated recombination and mutagenesis.    SOEing together tailor-made genes. Mol. Biotechnol. 3, 93-99.-   Horton, R. M., Cai, Z. L., Ho, S. N., and Pease, L. R. (1990). Gene    splicing by overlap extension: tailor-made genes using the    polymerase chain reaction. Biotechniques 8, 528-535.-   McCarrick, J. W., III, Parnes, J. R., Seong, R. H., Solter, D., and    Knowles, B. B. (1993). Positive-negative selection gene targeting    with the diphtheria toxin A-chain gene in mouse embryonic stem    cells. Transgenic Res. 2, 183-190.-   Yagi, T., Ikawa, Y., Yoshida, K., Shigetani, Y., Takeda, N.,    Mabuchi, I., Yamamoto, T., and Aizawa, S. (1990). Homologous    recombination at c-fyn locus of mouse embryonic stem cells with use    of diphtheria toxin A-fragment gene in negative selection. Proc.    Natl. Acad. Sci. U.S. A 87, 9918-9922.-   Yagi, T., Nada, S., Watanabe, N., Tamemoto, H., Kohmura, N., Ikawa,    Y., and Aizawa, S. (1993). A novel negative selection for homologous    recombinants using diphtheria toxin A fragment gene. Anal. Biochem.    214, 77-86.-   Zou, Y. R., Gu, H., and Rajewsky, K. (1993). Generation of a mouse    strain that produces immunoglobulin kappa chains with human constant    regions [see comments]. Science 262, 1271-1274.-   Zou, Y. R., Muller, W., Gu, H., and Rajewsky, K. (1994).    Cre-loxP-mediated gene replacement: a mouse strain producing    humanized antibodies. Curr. Biol. 4, 1099-1103.

1-32. (canceled)
 33. A hybridoma comprising a genetically-modifiedimmune cell having a vector comprising a fusion polynucleotide, saidfusion polynucleotide comprising a nucleic acid encoding animmunoglobulin molecule comprising two heavy chains and two lightchains, a nucleic acid encoding one detectable protein, and a nucleicacid encoding a flexible linker peptide located between the nucleic acidencoding the immunoglobulin molecule and the nucleic acid encoding theone detectable protein, and wherein said immune cell is capable ofexpressing one detectable protein fused with said immunoglobulinmolecule, with the flexible linker peptide between the immunoglobulinmolecule and the one detectable protein, wherein antibodies secreted bysaid genetically-modified immune cell comprise said one detectableprotein.
 34. A chimeric, detectably-labeled immunoglobulin moleculecomprising at least one detectable protein or peptide fused with thekappa immunoglobulin light chain, the lambda immunoglobulin light chain,an immunoglobulin heavy chain, or any combination thereof.
 35. Thechimeric, detectably-labeled immunoglobulin molecule of claim 34 whereinsaid at least one detectable peptide or protein is present at theC-terminus of the gene product of said fusion polynucleotide.
 36. Thechimeric, detectably-labeled immunoglobulin molecule of claim 35 whereina polynucleotide encoding said at least one detectable peptide orprotein present at the C-terminus of the gene product of said fusionpolynucleotide is located in exon G1.
 37. The chimeric,detectably-labeled immunoglobulin molecule of claim 35 wherein said atleast one detectable peptide or protein is present at the C-terminus ofthe gene product of said fusion polynucleotide with a flexible linkerpeptide therebetween.
 38. The chimeric, detectably-labeledimmunoglobulin molecule of claim 37 wherein a polynucleotide encodingsaid at least one detectable peptide or protein present at theC-terminus of the gene product of said fusion polynucleotide with aflexible linker therebetween is located in exon G1.
 39. The chimeric,detectably-labeled immunoglobulin molecule of claim 34 wherein saidimmunoglobulin heavy chain gene is selected from the group consisting ofIgG, IgM, IgD and IgA.
 40. The chimeric, detectably-labeledimmunoglobulin molecule of claim 34 comprising at least one detectableprotein or peptide in the heavy chain of said immunoglobulin molecule.41. The chimeric, detectably-labeled immunoglobulin molecule of claim 34comprising at least one detectable protein or peptide in the light chainof said immunoglobulin molecule.
 42. The chimeric, detectably-labeledimmunoglobulin molecule of claim 34 comprising at least one detectableprotein or peptide in the heavy chain and at least one detectableprotein or peptide in the light chain of said immunoglobulin molecule.43. The chimeric, detectably-labeled immunoglobulin molecule of claim 42wherein at least one said detectable protein or polypeptide is capableof quenching fluorescence.
 44. The chimeric, detectably-labeledimmunoglobulin molecule of claim 34 wherein said at least one detectableprotein or peptide is an autofluorescent protein or peptide, avisibly-detectable protein or peptide, an enzymatically active proteinor peptide, a protein or peptide capable of interacting with anothermolecule to produce a detectable product, or any combination thereof.45. The chimeric, detectably-labeled immunoglobulin molecule of claim 44wherein said at least one detectable protein is an autofluorescentprotein or peptide.
 46. (canceled)
 47. (canceled)
 48. The chimeric,detectably-labeled immunoglobulin molecule of claim 44 wherein said atleast one detectable protein is a combination of an autofluorescentprotein or peptide and an enzymatically-active protein or peptide. 49.The chimeric, detectably-labeled immunoglobulin molecule of claim 48wherein said at least one detectable protein is a combination of greenfluorescent protein and alkaline phosphatase.
 50. A method for producinga quantity of detectably-labelled polyclonal antibodies comprising thesteps of a) providing a genetically-modified mammal in accordance withclaim 1; b) immunizing said genetically-modified mammal with apreselected immunogen, wherein said genetically-modified mammalgenerates antibodies to said immunogen, wherein antibodies secreted byimmune cells of said genetically-modified mammal comprise said at leastone detectable protein or peptide; and c) isolating saiddetectably-labelled antibodies from said genetically-modified mammal.51. A method for producing a quantity of detectably-labelled monoclonalantibodies comprising the steps of a) preparing a genetically-modifiedmammal in accordance with claim 1; b) immunizing saidgenetically-modified mammal with a preselected immunogen, wherein immunecells of said genetically-modified mammal generate antibodies to saidimmunogen, wherein antibodies secreted by said immune cells comprisesaid at least one detectable protein or peptide; and c) immortalizingantibody-producing immune cells isolated from said genetically-modifiedmammal; d) selecting immortalized immune cells isolated from saidgenetically-modified mammal that secrete antibodies specific to saidimmunogen; and e) preparing a quantity of detectably-labeled monoclonalantibodies from said selected immune cells.
 52. A genetically-modifiednon-human mammal capable of producing a detectably-labeled antibody inresponse to immunization by an antigen, the genome of said non-humanmammal comprising at least one fusion polynucleotide comprising anucleic acid encoding at least one detectable protein or peptide and anucleic acid encoding an immunoglobulin molecule comprising two heavychains and two light chains, wherein an antibody secreted by an immunecell of said genetically-modified non-human mammal comprises said atleast one detectable protein or peptide.
 53. A chimeric,detectably-labeled immunoglobulin molecule comprising at least onefluorescent protein or peptide and at least one fluorescence-quenchingprotein or peptide fused with a component of said immunoglobulinmolecule independently selected from the group consisting of the kappaimmunoglobulin light chain, the lambda immunoglobulin light chain, animmunoglobulin heavy chain, and any combination thereof.
 54. Agenetically-modified non-human mammal whose genome comprises a vectorcomprising a fusion polynucleotide, said fusion polynucleotidecomprising a nucleic acid encoding an immunoglobulin molecule comprisingtwo heavy chains and two light chains, and a nucleic acid encoding atleast one detectable protein, wherein said non-human mammal is capableof expressing said at least one detectable protein fused with saidimmunoglobulin molecule wherein an antibody secreted by an immune cellof said genetically-modified non-human mammal comprises said at leastone detectable protein.
 55. The genetically-modified non-human mammal ofclaim 54 wherein said at least one detectable protein encoded by thefusion polynucleotide is present at the C-terminus of the gene productof said fusion polynucleotide.
 56. The genetically-modified non-humanmammal of claim 55 wherein said nucleic acid encoding said at least onedetectable protein present at the C-terminus of the gene product of saidfusion polynucleotide is located in exon G1.
 57. Thegenetically-modified non-human mammal of claim 54 wherein said at leastone detectable protein is present at the C-terminus of the gene productof said fusion polynucleotide with a flexible linker peptide locatedbetween the one detectable protein and the immunoglobulin molecule. 58.The genetically-modified non-human mammal of claim 54 wherein anantibody secreted by an immune cell of said genetically-modifiednon-human mammal comprises at least one detectable protein in the heavychain of said antibody.
 59. The genetically-modified non-human mammal ofclaim 54 wherein an antibody secreted by an immune cell of saidgenetically-modified non-human mammal comprises at least one detectableprotein in the light chain of said antibody.
 60. Thegenetically-modified non-human mammal of claim 54 wherein an antibodysecreted by an immune cell of said genetically-modified non-human mammalcomprises at least one detectable protein in the heavy chain and atleast one detectable protein in the light chain of said antibody. 61.The genetically-modified non-human mammal of claim 54 wherein the onedetectable protein is capable of quenching fluorescence.
 62. Thegenetically-modified non-human mammal of claim 54 wherein the onedetectable protein is an autofluorescent protein, a visibly-detectableprotein, an enzymatically active protein, or a protein capable ofinteracting with another molecule to produce a detectable product. 63.The genetically-modified non-human mammal of claim 62 wherein said onedetectable protein is an autofluorescent protein.
 64. Thegenetically-modified non-human mammal of claim 60 wherein said onedetectable protein is a combination of an autofluorescent protein and anenzymatically-active protein.
 65. The genetically-modified non-humanmammal of claim 64 wherein said one detectable protein is a combinationof green fluorescent protein and alkaline phosphatase.
 66. Agenetically-modified immune cell having a vector comprising a fusionpolynucleotide, said fusion polynucleotide comprising a nucleic acidencoding an immunoglobulin molecule comprising two heavy chains and twolight chains, a nucleic acid encoding at least one detectable protein,and a nucleic acid encoding a flexible linker peptide located betweenthe nucleic acid encoding the immunoglobulin molecule and the nucleicacid encoding the at least one detectable protein, and wherein saidimmune cell is capable of expressing at least one detectable proteinfused with said immunoglobulin molecule, with the flexible linkerpeptide between the immunoglobulin molecule and the one detectableprotein, wherein antibodies secreted by said genetically-modified immunecell comprise said one detectable protein.
 67. The genetically-modifiedimmune cell of claim 66 wherein said one detectable protein is presentat the C-terminus of the gene product of said fusion polynucleotide. 68.The genetically-modified immune cell of claim 66, wherein said nucleicacid encoding said one detectable protein present at the C-terminus ofthe gene product of said fusion polynucleotide is located in exon G1.69. The genetically-modified immune cell of claim 66 wherein an antibodysecreted by said immune cell comprises at least one detectable proteinin the heavy chain of said antibody.
 70. The genetically-modified immunecell of claim 66 wherein an antibody secreted by saidgenetically-modified immune cells comprises at least one detectableprotein in the light chain of said antibody.
 71. Thegenetically-modified immune cell of claim 66 wherein an antibodysecreted by said genetically-modified immune cells comprises at leastone detectable protein in the heavy chain and at least one detectableprotein in the light chain of said antibody.
 72. Thegenetically-modified immune cell of claim 66 wherein the one detectableprotein is capable of quenching fluorescence.
 73. Thegenetically-modified immune cell of claim 66 wherein the one detectableprotein is an autofluorescent protein or peptide, a visibly-detectableprotein or peptide, an enzymatically active protein or peptide, or aprotein or peptide capable of interacting with another molecule toproduce a detectable product.
 74. The genetically-modified immune cellof claim 73 wherein the one detectable protein is an autofluorescentprotein.
 75. The genetically-modified immune cell of claim 73 whereinsaid one detectable protein is a combination of an autofluorescentprotein and an enzymatically-active protein.
 76. Thegenetically-modified immune cell of claim 75 wherein said one detectableprotein is a combination of green fluorescent protein and alkalinephosphatase.
 77. The genetically modified non-human mammal of claim 62or the genetically modified immune cell of claim 73, or the chimericdetectably labeled immunoglobulin molecule of claim 44, wherein saidprotein capable of interacting with another molecule to produce adetectable product is selected from the group consisting of an intein, abiotin-binding subunit of streptavidin or avidin, a His tag, or achitin-binding domain, or any combination thereof, and wherein saidprotein capable of interacting with another molecule to produce adetectable product may also be used to facilitate purification of saiddetectable product.